U.S. patent application number 14/004145 was filed with the patent office on 2014-02-06 for rapid cell purification systems.
This patent application is currently assigned to Accelerate Technology Corporation. The applicant listed for this patent is Kenneth Robert Hance, Steven W. Metzger. Invention is credited to Kenneth Robert Hance, Steven W. Metzger.
Application Number | 20140038171 14/004145 |
Document ID | / |
Family ID | 46798781 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140038171 |
Kind Code |
A1 |
Metzger; Steven W. ; et
al. |
February 6, 2014 |
RAPID CELL PURIFICATION SYSTEMS
Abstract
Methods and systems for purifying cells and/or viruses are
provided. The sample is added to a well disposed in a medium. A
potential is applied across the medium to cause the contaminants to
enter one or more walls of the well, and retain the cells and/or
viruses in the well. The cells and/or viruses can be removed from
the well, and optionally adhered or fixed to a surface, or
detected. In one embodiment, the cells and/or viruses may be
retained in the well by embedding in the medium. The medium
including the embedded cells and/or viruses may be excised or
otherwise removed and transferred to a glass slide or other solid
surface.
Inventors: |
Metzger; Steven W.; (Tucson,
AZ) ; Hance; Kenneth Robert; (Tucson, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Metzger; Steven W.
Hance; Kenneth Robert |
Tucson
Tucson |
AZ
AZ |
US
US |
|
|
Assignee: |
Accelerate Technology
Corporation
Denver
CO
|
Family ID: |
46798781 |
Appl. No.: |
14/004145 |
Filed: |
March 7, 2012 |
PCT Filed: |
March 7, 2012 |
PCT NO: |
PCT/US12/28139 |
371 Date: |
October 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61449824 |
Mar 7, 2011 |
|
|
|
Current U.S.
Class: |
435/5 ;
435/173.9; 435/34 |
Current CPC
Class: |
C12M 47/12 20130101;
C12N 13/00 20130101; G01N 1/34 20130101; C12Q 1/24 20130101; B03C
2201/26 20130101; C12Q 1/02 20130101; C12N 7/02 20130101; G02B
21/361 20130101; G01N 33/491 20130101; B03C 5/005 20130101; C12N
2710/00051 20130101; C12N 7/00 20130101; B03C 2201/18 20130101 |
Class at
Publication: |
435/5 ;
435/173.9; 435/34 |
International
Class: |
C12N 13/00 20060101
C12N013/00 |
Claims
1. A method of purifying at least one of cells and viruses in a
sample comprising: adding the sample to a well disposed in a
medium; applying an electrical potential across the medium to cause
contaminants to enter said medium through one or more walls of said
well while retaining the at least one of the cells and/or viruses
in said well; and removing the at least one of the cells and
viruses from said well.
2. The method of claim 1, wherein said cells are
microorganisms.
3-7. (canceled)
8. The method of claim 1, wherein said medium comprises at least
one of a filter or a hydrogel.
9. (canceled)
10. The method of claim 8, wherein said hydrogel comprises at least
one of a polyacrylamide or agarose.
11. The method of claim 1, comprising placing a buffer in contact
with said medium.
12. The method of claim 11, wherein said buffer comprises histidine
and tris(hydroxymethyl)aminomethane.
13. The method of claim 1, further comprising immobilizing the at
least one of the cells and viruses.
14. The method of claim 13, wherein the at least one the of cells
and viruses are immobilized on a positively charged surface.
15. The method of any one of claim 1, further comprising detecting
the at least one of the cells and viruses.
16. The method of claim 1, further comprising adding a chemical
agent to the sample to increase a permeability of the medium to the
contaminants.
17. The method of claim 1, further comprising applying a tangential
flow to the medium to remove non-permeable contaminants from the
surface of the medium.
18. The method of claim 1, wherein the electrical potential
comprises an asymmetric alternating potential or a constant
potential.
19. The method of claim 1, further comprising reversing the
electrical potential to displace the at least one of the cells and
viruses from the surface of the medium.
20. The method of claim 1, wherein removing the at least one of the
cells and viruses from the well comprises removing at least a
portion of the medium including a wall of the well having the at
least one of the cells and viruses accumulated thereon.
21. The method of claim 20, further comprising fixing the portion
of the medium for staining or extracting molecular samples for
analysis.
22. The method of claim 1, further comprising providing a
localization device, wherein the localization device draws the at
least one of the cells and viruses to a discrete location in the
well thereby localizing the at least one of the cells and viruses,
and wherein the localization device is selected from at least one
of a non-conductive material, a conductive material, and a
discontinuous buffer system.
23-26. (canceled)
27. The method of claim 22, wherein the conductive material
comprises at least two electrodes.
28. The method of claim 27, wherein the at least two electrodes
comprises at least one sheet electrode.
29. (canceled)
30. The method of claim 13, wherein applying the electrical
potential and reversing the electrical potential are performed
iteratively.
31. The method of claim 1, wherein the electrical potential
comprises a field polarity and the filed polarity is switched
according to a programmed sequence.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a U.S. National Phase filing under
U.S.C. .sctn.371 of PCT/US2012/028139, filed Mar. 7, 2012, and
claims priority from U.S. Provisional Patent Application Ser. No.
61/449,824, filed Mar. 7, 2011, both of which are incorporated
herein by reference in their entirety.
FIELD
[0002] This disclosure relates to methods and systems for purifying
cells and/or viruses, particularly microorganisms in a sample,
particularly in preparation for diagnostics systems.
BACKGROUND
[0003] Diagnostic systems that detect cells and/or viruses are of
clinical and diagnostic interest. Detection of cells and/or viruses
is often prevented or complicated by the presence of contaminants
that interfere with collection or detection of the cells and/or
viruses. This may be particularly true for cells or viruses that
are adhered or fixed to a solid surface prior to detection.
[0004] Additionally, operator variability may adversely impact the
quality of specimen. Specimen quality is dependent on patient
factors including but not limited to differences between patients,
and the presence or absence of various interfering substances. In
many cases, the specimen is split and analyzed using various
diagnostic tests. Therefore, purifying samples reliably and
cost-effectively to remove inhomogeneities helps to improve the
likelihood of relevant statistical sampling of cells and/or viruses
therein.
SUMMARY
[0005] Methods and systems for purifying a microorganism are
provided. The sample is added to a well disposed in a medium. A
potential is applied across the medium to cause the contaminants to
enter one or more walls of the well, while the cells and/or viruses
are retained in the well. The cells and/or viruses can be removed
from the well, and optionally adhered or fixed to a surface, or
detected. In one embodiment, the cells and/or viruses may be
retained in the well by embedding in the medium. The medium
including the embedded cells and/or viruses may be excised or
otherwise removed and transferred to a glass slide or other solid
surface. The medium may then be cut or sectioned to correspond to
the respective wells. The medium is then dried, Gram stained, and
the cells/viruses detected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is a perspective view of an embodiment of a system
used to purify a microorganism.
[0007] FIG. 2A is a perspective view of an embodiment of a medium
in a cassette which may be used with the system of FIG. 1.
[0008] FIG. 2B is a partial perspective view of another embodiment
of a medium in a cassette which may be used with the system of FIG.
1.
[0009] FIG. 2C depicts a side view of the medium and system of FIG.
2B.
[0010] FIGS. 2D and 2E illustrate excision of a portion of the
medium of FIG. 2B.
[0011] FIGS. 2F, 2G and 2H depict the excised portion of the medium
of FIGS. 2D and 2E on a solid surface.
[0012] FIG. 2I depicts Gram stained microorganisms that may be
detected in accordance with the systems and methods described
herein.
[0013] FIG. 2J is a top view of a medium in a cassette which may be
used with the system of FIG. 1, wherein a localization device is
used to localize the cells and/or viruses.
[0014] FIG. 2K depicts Gram stained microorganisms that may be
detected in accordance with the systems and methods described
herein.
[0015] FIG. 3 is a side view of an embodiment of a custom
well-forming comb used to make wells in a medium.
[0016] FIGS. 4A and 4B are a perspective view of multiple flow cell
laminate assembly and a single flow cell cutaway view with
corresponding electrode and circuit details used to create a
potential across a medium.
[0017] FIG. 5 is a graph showing results of accumulated objects
(solid materials immobilized on a surface) over time for treated
compared to non-treated samples.
[0018] FIGS. 6A, 6B and 6C depict microscopic images of non-treated
samples over time.
[0019] FIGS. 7A, 7B and 7C depict microscopic images of treated
samples over time.
[0020] FIG. 8 is a side view of an embodiment of a circular well
formed in a medium with corresponding electrodes
[0021] FIG. 9 is a side view of an embodiment of a chamber formed
in the medium with an inlet and outlet port enabling sample
recirculation.
DETAILED DESCRIPTION
[0022] Described herein are various embodiments of systems and
methods for purifying cells and/or viruses in a sample. A sample
containing cells and/or viruses is added to a well disposed in a
medium. A potential is applied across the medium to cause
contaminants to enter the medium through one or more walls of said
well which retain the cells and/or viruses in the well. The cells
and/or viruses are then removed from the well. The cells and/or
viruses may also remain in or on the wall of the well, and/or the
wall/well may be excised for further analysis.
[0023] The systems and methods described herein may concentrate
cells and/or viruses from a low content specimen or sample in the
wells, thereby removing or reducing potentially interfering debris
and resulting in more readable specimens. For example, the
disclosed methods and systems may be used in testing of CSF
(cerebro-spinal fluid) specimens or other hypocellular specimens.
In such samples, bacterial organisms can be localized in 5.times.5
field of view capture areas (100.times. objective magnification) to
minimize time-consuming searching during microscopic examination. A
system having multiple wells may also be used to support parallel
processing of sample aliquots for concurrent analyses by multiple
downstream methods.
[0024] An exemplary embodiment of the system is depicted in FIG. 1.
FIG. 1 depicts a system 100 used to purify cells and/or viruses
with a cassette 106 configured to receive a medium. The cassette
106 includes a bottom plate 108 and sides 110. Negative electrode
102 and positive electrode 104 are operably connected to the medium
through a buffer (not shown) placed in a reservoir 112.
[0025] The sample is added to a well disposed in a medium,
preferably formed in the medium. In some embodiments, a plastic
well may also be disposed in the medium, in addition to well(s)
formed in the medium. An electrical potential is applied to the
well causing contaminant material to enter the medium while the
cells and/or viruses accumulate on the wall of the well. In some
embodiments, the cells and/or viruses may be localized on the wall
of the well. Cells and/or viruses remain in the well, thereby
purifying the sample. The sample may be mixed during or after a
time period of the applied electric field. In some embodiments, the
process can be repeated until separation of contaminants that
interfere with adhesion to a detection surface has been achieved.
The well can then be rinsed, and cells and/or viruses recovered. In
some embodiments, the wall of the well where the cells and/or
viruses have accumulated may be excised or otherwise removed from
the rest of the gel medium. Alternatively, the electrical potential
can be briefly reversed in polarity to displace the cells and/or
viruses from the wall prior to rinsing and recovery. Mixing,
applying a potential, and/or reversing polarity of applied field
can be performed iteratively to further purify the sample.
[0026] In some embodiments, the sample volume recovered is less
than, and sometimes substantially less than, the initial sample
volume in the wells. In one embodiment, a barrier, such as an
impermeable plastic sheet, is inserted into the wells and used to
reduce the volume in the wells, thereby further concentrating the
cells and/or viruses in the well and providing a reduced sample
volume for recovery.
[0027] Systems, including electrophoresis boxes and electrodes, can
be obtained from Thermo Fisher (Waltham, Mass.) under the
EC-Apparatus brand name (e.g., product number EC 250-90).
[0028] FIG. 2A depicts an embodiment of a medium in a cassette 106
comprising a bottom plate 108 and sides 110, which may be used with
the system 100. A medium 220 is disposed in the cassette 106 with a
plurality of wells 222 in the medium 220. A sample 224 is added in
some wells 222 with a pipette 226 comprising a pipette tip 228.
Although the medium is shown as a top load gel slab, media in other
forms, including but not limited to vertical gel slabs, can be
used.
[0029] FIGS. 2B-2H illustrate an embodiment where a medium 220 is
disposed in a cassette 106 and has a plurality of wells 222 in the
medium 220. A sample is added and a potential is applied between
negative electrode 102 and positive electrode 104 as described
elsewhere herein to cause contaminant material to enter the medium
while the cells 201 and/or viruses accumulate or become embedded on
the wall 223 of the well 222, depicted in FIG. 2C as concentrated
cells 202. As shown in FIGS. 2D-2E, a portion 230 of the medium
220, including at least a portion of the wells 222, is excised,
such as by cutting the medium along planes of excision defined by
lines A-A' and B-B', or otherwise removed from the medium. In some
embodiments, the excision can be robotically automated.
[0030] Following excision, the excised portion of the medium may be
fixed, for example, for staining or extracting molecular samples
for analysis. In some embodiments, and as shown in FIGS. 2F and 2G,
the excised portion 230 may be placed on a solid surface 232, such
as a glass slide, and the excised portion 230 may be sectioned at
each well 222 into sections 231. The sections 231 of excised
portion 230 may be dried, Gram stained, and detected as indicated
in FIGS. 2H-2I. In some embodiments, a prepared slide (e.g.,
wherein the sectioned, excised portion has been dried) can be
introduced into automated Gram staining equipment.
[0031] In some embodiments, the cells and/or viruses may be
localized on the wall of the well by, or with the help of, a
localization device. FIGS. 2J and 2K depict an embodiment
illustrating a localization device 240 and localized cells 241.
FIG. 2J is a top view of a gel medium that utilizes non-conductive
materials as a localization device to distort the electric field
resulting in localized concentration of cells and/or viruses, such
as microorganisms.
[0032] FIG. 2J illustrates an embodiment where the medium 220 is
disposed in the cassette 106 showing a plurality of wells 222,
including a plastic well 224, in the medium 220. The medium 220
includes a proximal end 220a and a distal end 220b. Disposed
between the wells 222 and the medium 220 is a localization device
240 comprising a non-conductive material including at least one
hole or aperture 240a. A sample is added and a potential is applied
as described elsewhere herein to cause contaminant material to
enter the medium while the cells and/or viruses accumulate or
become embedded on the wall 223 of the well 222. In this
embodiment, the sample flows through the aperture 240a in the
non-conductive localization device 240, thereby localizing the
cells and/or viruses that accumulate or become embedded on the wall
223 of the well 222, as described with reference to FIG. 2C. In
this embodiment, the non-conductive localization device 240
prevents or inhibits the sample from flowing anywhere but through
the aperture(s) 240a. In one embodiment, the non-conductive
material may be a plastic film. As described above with reference
to FIGS. 2D-2I, a portion of the medium 220, including at least a
portion of the wells 222, is excised or otherwise removed. The
excised portion 230 may be placed on a solid surface 232, such as a
glass slide, and may be dried, Gram stained, and detected. FIG. 2K
depicts Gram stained localized microorganisms 241 that have been
localized in accordance with the methods and systems described
herein.
[0033] While FIGS. 2J and 2K depict an embodiment of a system and
method for localization using a localization device 240 comprising
non-conductive materials, other methods and devices for
localization may also be used. In one embodiment, the localization
device is a conductive material, such as a metal or metal alloy
wire, that is embedded in or placed near a distal end 220b of the
medium 220 (i.e., downstream relative to the direction of
migration). When a potential is applied, the cells and/or viruses
localize on the wall of the well in a location corresponding to the
position of the conductive material. That is, where the conductive
material is a straight metal wire, the cells and/or viruses
localize on the wall of the well in a straight line corresponding
to the line of the metal wire. In another embodiment, the
localization device includes large and small (or discrete)
electrodes are used. For example, a large electrode, such as a
sheet electrode, may be placed at a proximal location relative to
the proximal end 220a of the medium 220. A small electrode may be
placed at a distal location relative to the distal end 220b of the
medium 220. When a potential is applied, the cells and/or viruses
localize on the wall of the well in a location corresponding to the
location of the small electrode. In still other embodiments, the
localization device is a discontinuous buffer system. In such a
system, the conductivity inside the well is different from the
conductivity outside the well. For example, where a well is made of
the medium, the conductivity of the sample and the conductivity of
the well are different. When a potential is applied, the cells
and/or viruses localize on the wall of the well due, at least in
part, to this conductivity difference.
[0034] Dyes can be used in samples to pre-label or added to provide
a tracking dye for purposes of a quantitative reference or sample
transfer quality control indicator. Examples of dyes include
colorants, bio-active adjuncts such as labeled antibodies, vital
stains, mortal stains (such as propidium iodide and the like).
Zwitterionic or neutrally charged dye molecules can be used to
monitor electro-osmotic flow.
[0035] The potential applied across the medium effective for
removal of contaminants can be applied for a variable time and is
dependent on the sample conductivity. For samples retrieved using
normal saline and having a conductivity near that of normal saline,
for example, the potential can be applied from 1 to 60 minutes.
[0036] In some embodiments, the method includes an asymmetric
alternating potential. In other embodiments, the potential is a
constant potential. In various embodiments, the applied potential
induces electro-osmotic flow that is used to remove contaminants
having a neutral charge. The potential can be reversed in polarity
to displace cells and/or viruses from the surface of the medium. In
some embodiments, the method includes applying a tangential flow
across the medium to remove non-permeable contaminants from the
surface of the medium. The tangential flow may be applied by
flowing the sample over the medium. The tangential flow may be
generated using additional buffer that is not the sample. The flow
can be continuously cycled over the medium.
[0037] When a sample is taken from a patient, there are various
components in the sample. For example, in a patient suffering from
pneumonia, a sample may include saline, anionic and cationic
species, pulmonary surfactants, bacteria, mucus, blood, host cells
such as white blood cells, and/or lung tissue cells. Mucus
components include, but are not limited to, mucoidal glycoproteins,
proteins, extra-cellular nucleic acids, F-actin, lysed white blood
cell fragments. Blood components may include, but are not limited
to, red cells, white cells, platelets, and plasma. Plasma
components may include, but are not limited to, sugar, fat, protein
and salt solution, platelets, blood clotting factors, sugars,
lipids, vitamins, minerals, hormones, enzymes, antibodies, and
other proteins including heme, albumins, immunoglobulins,
fibrinogens, regulatory proteins, lipoproteins (chylomicrons, VLDL,
LDL, HDL), transferrin, prothrombin, enzymes, proenzymes, residual
antibiotics used to treat the patient, and hormones. Lung tissue
components include host epithelial cells (intact or lysed). The
cells in the alveolar walls of the lung produce and secrete
pulmonary surfactant. Pulmonary surfactant is a mixture of
phospholipids and proteins. White blood cells may also be present
in lung samples. All the above components may be solubilized.
[0038] In some embodiments, the cells include blood cells, fungal
cells, bacterial cells, or microorganisms including parasites.
Examples of blood cells include red blood cells and white blood
cells. In some variations, the white blood cells can be
neutrophils.
[0039] In various embodiments, microorganisms can include bacteria,
fungi, algae, and protozoa. In one aspect, the microorganisms are
bacteria. The microorganisms can be pathogenic to humans and
animals. Suitable microorganisms include any of those well
established in the medical art and those novel pathogens and
variants that emerge from time to time. Examples of currently known
bacteria include, but are not limited to, genera such as Bacillus,
Vibrio, Escherichia, Shigella, Salmonella, Mycobacterium,
Clostridium, Cornyebacterium, Streptococcus, Staphylococcus,
Haemophilus, Neissena, Yersinia, Pseudomonas, Chlamydia,
Bordetella, Treponema, Stenotrophomonas, Acinetobacter,
Enterobacter, Klebsiella, Proteus, Serratia, Citrobacter,
Enterococcus, Legionella, Mycoplasma, Chlamydophila, Moraxella,
Morganella, and other human pathogens encountered in medical
practice. Included in the genera are various species. For example,
Klebsiella includes, but is not limited to, Klebsiella pneumoniae,
Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella
oxytoca, Klebsiella planticola, Klebsiella terrigena, and
Klebsiella ornithinolytica. Examples of viruses include
viroids.
[0040] Similarly, microorganisms may comprise fungi selected from
genera such as Candida, Aspergillus, and other human pathogens
encountered in medical practice. Viruses can be, but are not
limited to, orthomyxoviruses (e.g., influenza virus),
paramyxoviruses (e.g., respiratory syncytial virus, mumps virus,
measles virus), adenoviruses, rhinoviruses, coronaviruses,
reoviruses, togaviruses (e.g., rubella virus), parvoviruses,
poxviruses (e.g., variola virus, vaccinia virus), enteroviruses
(e.g., poliovirus, coxsackievirus), hepatitis viruses (including
hepatitis A, B, and C), herpesviruses (e.g., Herpes simplex virus,
varicella-zoster virus, cytomegalovirus, Epstein-Barr virus),
rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus
(e.g., rabies virus), retroviruses (including HIV, HTLVI and II),
papovaviruses (e.g., papillomavirus), polyomaviruses,
picornaviruses, and the like.
[0041] The methods and systems described herein can be used to
identify host cells harboring viruses. The cells are first
purified, and subsequently the cells are manipulated to either
produce viruses, or to identify nucleic acids in the cells.
[0042] The sample can be obtained from any number of sources,
including, but not limited to, bodily fluids (including, but not
limited to, blood, urine, serum, lymph, saliva, anal and vaginal
secretions, perspiration, peritoneal fluid, pleural fluid,
effusions, ascites, and purulent secretions, lavage fluids, drained
fluids, brush cytology specimens, biopsy tissue, explanted medical
devices, infected catheters, pus, biofilms and semen) of virtually
any organism, including mammalian samples and human samples, as
well as environmental samples (including, but not limited to, air,
agricultural, water and soil samples). In addition, samples can be
taken from food processing, which can include both input samples
(e.g., grains, milk or animal carcasses), samples in intermediate
steps of processing, as well as finished food ready for the
consumer. The method can be used for veterinary applications. The
methods can be also used for the analysis of milk in the diagnosis
and treatment of mastitis, and the analysis of respiratory samples
for the diagnosis of bovine respiratory disease. Furthermore, the
methods provide for the rapid detection of the presence of
potential biological warfare agents in a sample.
[0043] Samples can range from less than a milliliter to up to a
liter for certain respiratory lavage fluids, and can further range
in bacterial concentration from less than one bacterium to greater
than 10.sup.9 bacteria per milliliter. Furthermore, the sample can
be present in blood, urine, sputum, lavage fluid or other medium.
The sample can be concentrated prior to using the described methods
for purifying cells and/or viruses from the sample. Sample
concentration both concentrates the sample so that bacteria that
are present in small numbers can all be effectively introduced into
the system and adequately sampled, as well as so the background
liquid medium can be normalized, or in some cases eliminated or
reduced, to have consistent properties upon introduction to the
system. Sample concentration can be performed by centrifugation,
combining samples, removing solvents, and the like. It should be
noted, however, that certain samples provided in the description
can be used without concentration or other modification.
[0044] The rapid detection of various cells and/or viruses is
useful for a patient suffering from various diseases and disorders.
For example, pneumonia can result from a variety of causes,
including infection with bacteria, viruses, fungi, or parasites, as
well as chemical or physical injury to the lungs. However, some
samples of cells and/or viruses contain contaminants that interfere
with their detection. Purification of a microorganism (or virus or
other cell), and detection of the type and amount of a
microorganism (or virus or other cell) present in a sample, are
helpful to diagnose and treat a patient effectively.
[0045] In other embodiments, the cells are selectively lysed. For
example in the case of intracellular targets, the mammalian cells
can be lysed, releasing intracellular microorganisms prior to,
during, or after the purification described herein.
Contaminants
[0046] Contaminants are removed from the sample into the medium.
Contaminants that can be removed include ionic species, including,
but not limited to, mono or divalent cations and anions, released
intracellular materials, phospholipids, extracellular proteins,
mucins, pulmonary surfactants, mucus plugs, pus, glycoproteins, and
nucleic acids. Removing contaminants avoids other time intensive
preparation steps such as vortexing and centrifugation. In various
aspects, the removed contaminants interfere with cells and/or virus
surface immobilization, detection, and imaging. Cells and/or
viruses remain in the well, and can be recovered. In certain
aspects of the purification methods, certain components, such as
cellular membrane fragments and larger cellular fragments, are not
removed from the sample.
Medium
[0047] Organogels, xerogels, and aerogels may also be utilized as
the medium. Aerogels include, but are not limited to, silica
aerogel, carbon aerogels, alumina, cadmium, and selenide aerogels.
Organic aerogels, such as SEAgel, are made of agar. Aerogels made
of chalcogens such as sulfur, selenium, and other elements may also
be of utility.
[0048] In various embodiments, the medium is a hydrogel. In some
embodiments, hydrogels are a network of polymer chains that are
hydrophilic. Hydrogels can be highly absorbent natural or synthetic
polymers, and in some instances can contain over 99% water. In
general, hydrogels are solid, yet porous media.
[0049] The concentration of the hydrogel affects the migration
speed of the contaminants through the hydrogel. Increasing the
concentration of the hydrogel decreases the pore size within the
hydrogel. Additionally, contaminants with smaller molecules move
faster and migrate further than contaminants with larger
molecules.
[0050] The charge of the hydrogel also affects the migration speed
of the contaminants through the hydrogel. Each contaminant molecule
migrates to the electrode that carries a charge opposite of that of
the contaminant molecule. Most biological materials have a net
negative surface charge. Some have a net positive charge if the
material has an excess of amines or other positively-charge
moieties exposed to the surface. The charge is considered neutral
if it is a balance of positive and negative, or uncharged, such as
complexes coated with neutral materials that envelope and screen
charged materials within. The uncharged material will migrate in
the direction of electro-osmotic flow, if present.
[0051] The pH of the hydrogel also affects the migration speed of
the contaminants and the targets. In some embodiments, the pH is
selected to enhance mobility of the contaminants relative to the
cells and/or viruses. In some embodiments, a pH may be selected
such that the cells and/or viruses are substantially near the
isoelectric point, minimizing the cells' and/or viruses' mobility
relative to the contaminants. In other embodiments, the pH may be
selected to be substantially different from the isoelectric point
such that the direction of the cells' and/or viruses' mobility is
reversed relative to the contaminants.
[0052] In some embodiments, a medium contains nutrients that
promote the viability of the cells and/or viruses.
[0053] Media used in the systems described can separate contaminant
molecules based on both their size and their charge.
[0054] The hydrogel's porosity is directly related to the
concentration of agarose in the medium. Various levels of effective
viscosity can be selected, depending on the experimental
objectives.
[0055] Examples of hydrogels are alginates, as disclosed in
Gadkari, 2007, "Optimal hydrogels for fast and safe delivery of
bioactive compounds", Thesis of Drexel University;
ethyl-vinyl-acetate copolymer as disclosed in U.S. Pat. No.
3,854,480; esters of hydantoic acid as disclosed in U.S. Pat. No.
3,792,081, olefin saturated polyester 500-8000, polyethylene glycol
(PEG) 200-1500, ethyl-vinyl-acetate copolymer 20-40% VA (20-30K),
chlorinated polyethylene 25-45% Cl-- (20-30K), ethyl-ethylacrylate
copolymer 20-40% EA (20-30K), and ethylene vinyl chloride copolymer
25-45% Cl-- (20-30K) as disclosed in U.S. Pat. No. 3,938,515;
methyl-methacrylate copolymer and glyceryl-methyacrylate copolymer
as disclosed in U.S. Pat. No. 3,957,362; ethylene-vinyl-actetate
copolymer 4-80% VA (20-30K) as disclosed in U.S. Pat. No.
4,069,307; polysiloxanes as disclosed in U.S. Pat. No. 4,136,250;
hydrophilic dihydroxyalkyl acrylate and insoluble copolymer as
disclosed in U.S. Pat. No. 4,267,295; cellulose triacetate as
disclosed in U.S. Pat. No. 4,220,152; acrylamide, vinylpyrrolidone,
and polyethyleneoxide diol as disclosed in U.S. Pat. No. 4,423,099;
poly-amino acid homopolymers and copolymers as disclosed in U.S.
Pat. No. 4,351,337; poly-glutamic acid ethyl-glutamate copolymer
(5-50% GA, 80-500 KDa) as disclosed in U.S. Pat. No. 4,450,150;
polyoxyethlyene-polyoxypropylene copolymer thermoset as disclosed
in U.S. Pat. No. 4,478,822; vinyl cross-linked copolymers of
insoluble and soluble monoolefinic esters as disclosed in U.S. Pat.
No. 4,548,990; copolymers with N-vinyl-2-pyrrolidone and
methacrylates as disclosed in U.S. Pat. No. 4,693,884;
polyanhydride as disclosed in U.S. Pat. No. 4,657,543; colpolymer
of poly(alkylene oxide) and cyclic ester of alpha hydroxy acid
(glycolide) as disclosed in U.S. Pat. No. 4,882,168;
polyacrylonitrile-nitric acid copolymer as disclosed in U.S. Pat.
No. 5,218,039; N-morpholinoethyl methacrylate and 2-hydroxyethyl
methacrylate copolymer as disclosed in U.S. Pat. No. 4,857,313;
crosslinked copolymers of vinyl pyrrolidone and allylamine as
disclosed in U.S. Pat. No. 4,772,484; water soluble polyacetals
having molecular weights from about 5,000-30,000 as disclosed in
U.S. Pat. No. 4,713,441; thermoplastic hydrogels of polyvinyl
pyrrolidone (PVP) and polyvinyl acetate (PVA), and gelatin as
disclosed in U.S. Pat. No. 5,002,792; alginic acid with Ca++, Ba++
or Zn++, pectic acid with Ca++, Ba++ or Zn++, hyaluronic acid with
Ca++, Ba++ or Zn++, polyglucuronic acid with Ca++, Ba++ or Zn++,
polymanuronic acid with Ca++, Ba++ or Zn++, polygalacturonic acid
with Ca++, Ba++ or Zn++, polyarabinic acid with Ca++, Ba++ or Zn++,
and kappa-carrageenan with Ca++, Ba++ or Zn++, as disclosed in U.S.
Pat. No. 5,089,606; charged side-chain polyphosphazenes with Ca++
cross-linking as disclosed in U.S. Pat. No. 5,149,543;
carboxymethylcellulose as disclosed in U.S. Pat. No. 5,208,037;
agarose as disclosed in U.S. Pat. No. 3,961,628; polyacrylamide as
disclosed in U.S. Pat. No. 6,391,937; pluronic 127,
N-isopropylacrylamide (NiPAM); and blends (block co-polymer, etc.)
of all the above listed hydrogels.
[0056] Agarose is a linear polymer, made up of the repeating
monomeric unit of agarobiose. Agarobiose is a disaccharide made up
of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose pectin or
sulfonated agarose can be used as the hydrogel. Agarose can be
obtained from Lonza (Rockland, Me.) under the brand name
SeaKem.TM.. In certain embodiments, the concentration of the
agarose gel for effectively removing contaminants is from 0.1-2.0%
w/v.
[0057] Purified agarose hydrogels may be purchased for use in the
described method. An example of a commercial purified hydrogel can
be obtained from Invitrogen (Carlsbad, Calif.) under the brand name
E-Gel.RTM. EX Starter.
[0058] Polyacrylamide is a polymer (--CH.sub.2CHCONH.sub.2--)
formed from acrylamide subunits. It can be synthesized as a simple
linear-chain structure or cross-linked, typically using
N,N'-methylenebisacrylamide. In the cross-linked form,
polyacrylamide is highly water-absorbent, forming a soft gel.
Polyacrylamide can be obtained from BioRad (Hercules, Calif.).
[0059] Purified polyacrylamide hydrogels may be purchased for use
in the described method. An example of a commercial purified
hydrogel can be obtained from BioRad (Hercules, Calif.).
[0060] Preconditioning of a medium can be performed.
Preconditioning of a medium is often done to remove impurities
found in the medium. For example, providing a potential across a
hydrogel helps mobile impurities to migrate outside of the
hydrogel. The potential can be, for example, 50V, 75V, 100V, 150V,
200V, 250V, 300V, 350V, 400V or 500V. In various embodiments, the
potential can be provided for a period of time, such as at least 2
minutes, at least 5 minutes, at least 10 minutes, at least 15
minutes, at least 30 minutes, at least 45 minutes, at least 60
minutes, at least 120 minutes, or at least 180 minutes.
[0061] In various embodiments, the medium can be a filter. Examples
of filters include those available from Pall Corporation (Port
Washington, N.Y.), such as hydrophilic polypropylene, ahydrophilic,
low binding material with pore sizes of 0.2 .mu.m and 0.45 .mu.m;
polytetrafluoroethylene (PTFE), a hydrophobic, high binding
material with pore sizes of 0.2 .mu.m, 0.45 .mu.m, 1 .mu.m, 2 .mu.m
and 3 .mu.m; glass fiber, a hydrophilic, moderate binding material
with a pore size of 1 .mu.m; nylon, a hydrophilic, low binding
material with pore sizes of 0.2 .mu.m and 0.45 .mu.m;
polyvinylidene fluoride (PVDF), a hydrophilic, low binding material
with pore sizes of 0.2 .mu.m and 0.45 .mu.m; PES (Supor.RTM.), a
hydrophilic, low binding material with pore sizes of 0.1 .mu.m, 0.2
.mu.m, 0.45 .mu.m, and 0.8 .mu.m; vinyl/acrylic copolymer, a
hydrophobic material that may be used for air sampling and has pore
sizes of 0.45 .mu.m and 0.8 .mu.m; polyvinyl chloride (PVC), which
may also be used for air sampling and has pore sizes of 5 .mu.m;
hydrophilic mixed cellulose esters, a high binding material with a
pore size of 0.45 .mu.m; hydrophilic acrylic copolymer, which may
be used as a pre-filter on a support and has pore sizes of 0.2
.mu.m, 0.45 .mu.m, 0.8 .mu.m, 1.2 .mu.m, 3 .mu.m, and 5 .mu.m; and
nitrocellulose, a high binding material with a pore size of 0.2
.mu.m. Examples of filters available from Millipore (Billerica,
Mass.) include PTFE (LCR), a hydrophilic, moderate binding material
with pore sizes of 0.2 .mu.m and 0.45 .mu.m; PVDF (Durapore.TM.), a
hydrophilic, low binding material with pore sizes of 0.2 .mu.m and
0.45 .mu.m; PTFE (Fluoropore.TM.), a hydrophilic, low binding
material with pore sizes of 0.2 .mu.m and 0.45 .mu.m; nylon, a
hydrophilic, low binding material with pore sizes of 0.2 .mu.m and
0.45 .mu.m; glass fiber, a hydrophilic, moderate binding material
with a pore size of 1 .mu.m; and hydrophilic mixed cellulose
esters, a high binding material with exemplary pore sizes of 0.2
.mu.m, 0.45 .mu.m, and 0.8 .mu.m. Filters can have pore sizes of
greater than or equal to about 0.01 .mu.m, 0.05 .mu.m, or 0.1
.mu.m, 0.2 .mu.m, 0.4 .mu.m, 0.6 .mu.m, 0.8 .mu.m, 1.0 .mu.m, 1.5
.mu.m, 2.0 .mu.m, 2.5 .mu.m, 3.0 .mu.m, 4.0 .mu.m, or 5.0 .mu.m.
Filters can have pore sizes of less than or equal to about 5.0
.mu.m, 4.0 .mu.m, 3.0 .mu.m, 2.5 .mu.m, 2.0 .mu.m, 1.5 .mu.m, 1.0
.mu.m, 0.8 .mu.m, 0.6 .mu.m, 0.4 .mu.m, 0.2 .mu.m, 0.05 .mu.m, or
0.01 .mu.m.
[0062] In various embodiments the method includes adding a chemical
agent to the medium to increase the permeability of the medium
and/or increase the ability of the contaminant to enter the
medium.
[0063] Examples of chemical agents include reducing agents,
including, but not limited to, dithiothreitol (DTT),
tris(2-carboxyethyl)phosphine (TCEP), and mercaptoethanol reducing
agents; denaturing agent using surfactants, including, but not
limited to, sodium lauryl sulfates, non-ionic surfactants such as
Triton X-100, Tween-20, or chaotropic agents, including, but not
limited to, urea, thiourea, or guanidinium chloride; chelating
agents that can coordinate molecules such as calcium, magnesium,
and other divalent and trivalent ions (including metal ions),
including ethylenediaminetetraacetic acid (EDTA) and ethylene
glycol tetraacetic acid (EGTA); cleavage agents including
proteases, nucleases, glyconases, lipases; and excipients such as
polyethylene glycol. In some embodiments a combination of one or
more chemical agents can be utilized.
[0064] Viscous gels include cellulose ethers (such as hydroxylethyl
cellulose or Methocel.TM. (Dow (Midland, Mich.)) and soluble
polymer viscosity modifiers (such as polyethylene glycol,
polyvinylpyrrolidone, dextrans, pluronic surfactants, and
alginates). In a viscous gel, the pore size is not defined. The
separation is based on retarded flow of the cells in the viscous
medium.
[0065] In some embodiments, agents can be added to or used to treat
the medium to control electroosmotic flow. In some embodiments, it
may be desirable to increase or decrease electroosmotic flow.
Sample Mixing
[0066] To analyze a representative sample, the sample should be
substantially uniform. In some embodiments, the homogenization of a
sample can be done by a sample mixing or stirring step. Mixing the
sample acts to re-suspend any caked material formed on the walls of
the well.
[0067] In various embodiments, the method includes mixing a sample
using a pipette tip. See, for example, the pipette tip 228 in FIG.
2A. The sample is passed through the narrow opening of the pipette
to shear and homogenize the sample.
Buffer Solutions
[0068] In various embodiments, the method includes placing a buffer
in contact with the medium.
[0069] In some embodiments, the mixing parameters of the buffer are
designed to maximize the removal of debris and non-target
material.
[0070] In some embodiments, the buffer can be replenished to
prevent accumulation of undesirable electrophoresis products. For
example, undesirable effects pH gradients generated at the cathode
and anode and in proximity to the sample can be substantially
minimized by buffer replenishment or replacement, potentially using
continuous flow.
[0071] In various embodiments, electrophoretic buffers utilize
pairs of redox mediators. In certain embodiments, these redox
mediators facilitate low voltage electrophoresis that permits cell
viability to be maintained. These redox mediators may also enable
the use of electrode materials that have limited utility in high
voltage electrophoresis (for example, indium tin oxide, "ITO"
electrodes). In addition, these redox mediators find use in "closed
systems" (i.e., systems not open to the atmosphere). In closed
systems, bubble formation and generation of other reactive species
during the electrophoresis step, which can cause a number of
problems, is prevented, and closed systems also help to prevent the
exposure of the technician to potentially infectious samples, as
well as reducing problems associated with discarding biological
samples
[0072] In some embodiments, the buffer is placed in a reservoir in
contact with the medium. In various embodiments, the medium is not
submerged in the buffer.
[0073] Buffers include, for example, various electrophoresis
buffers including zwitterionic buffers, neutral buffers such as
phosphate-buffered saline (PBS), lower or higher pH buffers, and
hypotonic or hypertonic buffers. In some embodiments, borate and
other selected ions and counter-ions are included to facilitate
effective electrophoresis.
[0074] In some embodiments, the buffer solution includes histidine
and tris(hydroxymethyl)aminomethane. Histidine has low
conductivity. Tris(hydroxymethyl)aminomethane has some conductivity
but has low mobility. Histidine has pKa values close to
physiological values providing adequate buffering capacity.
Tris(hydroxymethyl)aminomethane can be obtained from Sigma-Aldrich
(St. Louis, Mo.) as Trizma.RTM. base (Sigma, T1503).
[0075] In some embodiments, the sample (in 150 mM NaCl) is desalted
to remove cationic and anionic species that may interfere with
subsequent analysis. In some embodiments, desalting allows
successful concentration and capture of the microorganism.
[0076] Electrophoretic mobility can be buffer dependent due to zeta
potential variability with salt concentration, valency of salts
present in the buffer, and the pH of the buffer. Bacteria can lose
charge as the concentration of salt increases or as the pH is
lowered below a certain pH, for example, pH 5.0. Divalent and
trivalent salts are more effective quenchers than monovalent salts.
For example, CaCl.sub.2 is more effective than NaCl to quench a
charge. Certain agents such as chelators, including, but not
limited to, ethylenediaminetetraacetic acid (EDTA) and ethylene
glycol tetraacetic acid (EGTA), both available from Sigma-Aldrich,
can be used to control the concentration of charged species in the
sample.
Wells
[0077] As shown in FIG. 2A, one or a plurality of wells can be
formed in the medium. The wells are molded into the gel. For
example, a custom well-forming comb can be used to create the
appropriate well shape. Wells include side-walls that can be
substantially vertical or diagonal. In various embodiments, the
method includes wells that are non-rectangular shaped. In various
embodiments, the wells are substantially chamfered to eliminate
sharp edges in the well, enhancing target recovery. In some
embodiments, the wells can hold various sample sizes. In various
embodiments, the wells can hold from 10 .mu.L to 500 .mu.L. In some
embodiments the wells are 5 to 250 mm wide. Multiple wells can be
used for a sample. As illustrated in FIG. 8, a well 822 in medium
820 may be circular, surrounding an electrode such as cathode 804,
and the counter electrodes may surround the well, such as the
illustrated anode ring 802. In a circular well configuration, a
sample placed in well 822 surrounds the cathode 804. In such an
embodiment, a run buffer sheath may flow over cathode 804 to remove
electrode byproducts during electrophoresis.
[0078] FIG. 3 depicts a side view of an embodiment of a custom
well-forming comb 300. A comb body 302 has a plurality of
well-forming teeth 304 connected to the comb body 302 by one edge
306. The sides of the teeth 308 form the side-walls of the
non-rectangular shaped wells in a medium. In this embodiment the
wells are triangular-shaped. The comb 300 is sized and shaped to
fit the medium in a cassette, such as the cassette shown in FIG.
2A. In the illustrated embodiment, the comb 300 has six
well-forming teeth 304, but could have more or less teeth depending
on the size of the cassette.
[0079] In a rectangular or square bottomed well, sample solution
can wick up the walls of the well. In a triangular shaped well, the
sample solution does not tend as strongly to wick up the walls,
making it easier to remove the microorganism from the well. In some
embodiments, the triangular shaped well is narrowest at the bottom
and widest at the top of the well. The well-forming teeth 304 shown
in FIG. 3 create a pattern of triangular shaped wells in the
medium. In other embodiments, the wells may be round-bottomed
wells.
[0080] The samples can have high solids (e.g., from 1%-50%
weight/volume of solid components). Minimizing the well width
minimizes the caking of the solids on the well walls. In some
embodiments, the well is 0.0025 inches wide at the widest
point.
Chambers
[0081] In various embodiments, the method uses a system 900 wherein
one or a plurality of chambers 953 can be formed in a medium 920.
The chambers 953 are molded into the gel 920 and have an inlet and
outlet port (954 and 955) as shown in FIG. 9. Inlet and outlet
ports 954 and 955 are connected by tubing 956 for recirculating a
sample through a chamber 953, such as by a peristaltic pump. The
chambers may be submerged or partially submerged in buffer, and
electrical potential is applied to the system orthogonally to the
direction of the recirculating flow of sample.
Electrodes
[0082] In various embodiments of the method, an electrode or a
plurality of electrodes may be contained within the well or
chamber. Additionally, in various embodiments, the electrode or
plurality of electrodes may be in contact with the medium or
separated a distance from the medium. The electrode or plurality of
electrodes may be connected to the medium using salt bridges,
buffer, redox mediators, or other conductive charge transfer
methods used by those skilled in the art or familiar with
techniques used in applications for establishing faradaic current.
In some embodiments the electrodes are in physical contact with the
chamber walls.
[0083] In various embodiments, conductive materials may be utilized
to distort the electric field resulting in localized concentration
of cells and/or viruses. Electric field distortion may utilize
material conductivity differences to accomplish the said
localization.
Immobilization of the Cells and/or Viruses
[0084] In various embodiments, the method includes immobilizing the
microorganism. Cells and/or viruses are immobilized by various
filters that exclude the targets (microorganism) from penetrating,
for example, tube walls, microchannels (horizontal or vertical), or
any geometry that uses a capture surface (specific or nonspecific),
mazes, fluidic dead space (eddy cul-de-sacs), and microwells of
approximate cellular scale.
Detection Surface
[0085] In some embodiments, the method includes immobilization of
cells and/or viruses on a positively charged surface. For example,
cells and/or viruses can be immobilized by a positively charged
detection surface. In other embodiments, the cells and/or viruses
may be immobilized by embedding in the medium.
[0086] Detection surfaces are disclosed in, for example, U.S. Pat.
No. 6,844,028, incorporated by reference herein in its entirety.
Detection surfaces can include those coated with poly-L-lysine,
polyethylenimine, or other cationic polymers. Additionally,
detection surfaces can include hydrophobic surface coatings.
[0087] After the contaminants are removed from the sample by the
medium, the microorganism can be detected by a system. In some
embodiments, the system is an optical sensing system. In some
embodiments, the system is a microscope.
[0088] In some embodiments, the system is an automated system.
[0089] In various embodiments, the sequential or simultaneous use
of a plurality of electrophoresis electrodes allows
multidimensional electrophoresis, i.e., the solution may be
targeted, "mixed," or "stirred" in the vicinity of a detection
surface to further increase the kinetics of binding. For example,
polarities can be reversed to allow cells and/or viruses that may
not have bound to the detection surface to travel back "over" the
surface, resulting in increased binding. Also, electrodes may be
located and field polarity switched according to a programmed
sequence so as to provide agitation in two dimensions of a plane,
or in three dimensions.
Detection of the Microorganism
[0090] In various embodiments, the method includes detection of the
microorganism. In general, biosensor devices are designed to fit
into a detection unit, and generally utilize a number of
components, which can either be "on-chip" (e.g., part of a
biosensor cartridge) or "off-chip" (where some of the components
are part of separate device or devices into which the biosensor
cartridge fits). These components include, but are not limited to,
one or a plurality (e.g., an array) of detection surface(s),
concentration modules (which, as outlined herein, frequently are
configured with the detection surface(s)), detection modules
(again, frequently configured with the detection surface(s)), input
and output ports, channels, pumps, mixers, valves, heaters, fluid
reservoirs (including sample reservoirs, reagent reservoirs, and
buffer reservoirs), concentration controllers (e.g., in the case of
electrophoresis, electrical controllers), and data collection and
analysis (e.g., computer) components.
[0091] An example of a microorganism diagnostic system is described
in U.S. patent application Ser. No. 10/888,828 filed Jul. 8, 2004,
issued as U.S. Pat. No. 7,687,239, and U.S. application Ser. No.
11/303,803, filed Dec. 16, 2005, issued as U.S. Pat. No. 7,341,841,
both of which are incorporated herein by reference in their
entirety.
[0092] Low levels of cells and/or viruses can be detected with this
method. Cells can be measure in terms of cells per mL, colony
forming units (CFU, or units) per mL for fungi and/or bacterial
microorganisms, and viruses can be measured in particles per mL or
plaque forming units per mL (PFU). Levels of cells and/or viruses
are described in units per volume, typically per mL volume. Those
skilled in the art understand the specific units are typically
reported as appropriate for a given target. For exemplary purposes,
the concentration ranges below are reported in generic units per
mL. For example, levels of 0.1 to 1.times.10.sup.8 units/mL can be
detected. In various embodiments, cells and/or viruses of levels
less than 5.times.10.sup.8 units/mL, 3.times.10.sup.8 units/mL,
1.times.10.sup.8 units/mL, 0.8.times.10.sup.8 units/mL,
0.6.times.10.sup.8 units/mL, 0.4.times.10.sup.8 units/mL,
0.2.times.10.sup.8 units/mL, or 0.1.times.10.sup.8 units/mL, can be
detected.
EXAMPLES
[0093] The following examples are provided for illustration
purposes and are not intended to limit scope. Other variants will
be readily apparent to one of ordinary skill in the art and are
encompassed by the appended claims.
[0094] The example described below anticipates a wide range of
specimen variability, first homogenizing the specimen, then
sampling the specimen, and then purifying the sample to remove
debris and other interfering materials by placing the sample in a
medium containing a well and applying a potential laterally across
the medium to retain cells and pass contaminants into the
medium.
Example 1
Purification of Bacteria Cells from Respiratory Specimens
Gel Preparation
[0095] 10 grams of agarose powder (SeaKem, LE Agarose) was mixed
with 1 L of buffered solution containing 100 mM histidine (Sigma,
H8000) and 2.5 mM Trizma.RTM. base (Sigma, T1503). The final
concentration of agarose slurry was 1.0% (w/v). The solution was
boiled to melt the agarose powder and the molten agarose was stored
in liquid form at 40.degree. C. until use.
Gel Casting
[0096] Those familiar in the art of gel slab electrophoresis
recognize that solid inserts or combs are routinely used to create
a void volume in a gel slab that is later utilized for sample
loading. Gel electrophoresis combs are generally nominally 1-2 mm
thick, capable of holding nominally 100 .mu.L of sample volume. In
this example, a custom equilateral V-shaped well was used. The well
had sides 1 cm long and a thickness of nominally 0.6 mm (0.025'').
The comb was inserted into a gel box container (E-C Apparatus, EC
250-90) and the box filled with the molten agarose submerging a
portion of the comb. The agarose was allowed to cool to room
temperature forming an agarose gel. The comb was removed from the
solidified agarose and the void volume of the comb formed a well in
the material. The V-shaped well enabled facile recovery of the
sample volume from the well, described in further detail below.
Pretreatment of the Agarose Gel Medium
[0097] The gel box containing the agarose gel medium having
triangular wells was placed in an electrophoresis apparatus and
then submerged in a run buffer containing 100 mM histidine and 2.5
mM Trizma.RTM. base. A 250 volt potential was applied for 1 hr. The
applied potential yielded 22 mA of current. The pretreated gels
were removed from the electrophoresis apparatus and transferred to
a closed container and stored submerged in fresh run buffer until
use.
Specimen Homogenization
[0098] A remnant specimen having a known level of bacteria was
homogenized by pouring into a syringe connected to 0.02'' (0.5 mm)
inner diameter PEEK tubing and forcing through the PEEK tubing 10
times at a flow rate of approximately 0.1 mL/sec to liquefy the
specimens. The specimen was then filtered through 5 .mu.m track
etch polycarbonate filters (SPI Pore, E5013-MB). A 1 mL sample
aliquot of the specimen was processed as described below. An
aliquot of the specimen was also reserved as a control.
[0099] A control or a known clinical sample (e.g., with a known
concentration of bacteria) can be compared to the unknown
sample.
Assessment of the Sample
[0100] The sample was diluted to a final nominal bacterial
concentration of 1.5.times.10.sup.3 CFU/mL. 50 .mu.L of the diluted
sample was plated in triplicate on Mueller Hinton Agar (MHA) and
placed in the incubator overnight. The number of colonies counted
on the overnight incubated plates divided by the plated volume and
multiplied by dilution factor yielded the actual number of input
Klebsiella oxytoca bacteria in CFU/mL.
[0101] The sample was diluted 10-fold and the optical density read
was acquired at 625 nm to assess the amount of particulate debris
in the sample.
Sample Loading
[0102] The pretreated gels were placed in the gel box and
apparatus, patted dry, and excess liquid was removed from the
triangular wells using 0.2 mm thick flat capillary plastic pipette
tips (Fisher 07-200-519). The well was filled with a 20 .mu.L
sample of the homogenized specimen.
Sample Treatment
[0103] Histidine/Tris run buffer was added to the apparatus so that
the liquid level was below the top of the gel slab. The sample was
electrophoresed for 5 minutes at 250 volts and the samples were
hydrodynamically sheared by pipetting the sample volume up and down
5.times. using a capillary pipette tip. The samples were
electrophoresed again for 5 minutes at 250 volts and the samples
then hydrodynamically sheared by pipetting the sample volume up and
down 5.times. using a capillary pipette tip.
Post-Treatment Assessment of Spiked Sample
[0104] The treated sample was diluted to a final concentration of
1.5.times.10.sup.3CFU/mL. 50 .mu.L of the diluted sample was plated
in triplicate on Mueller Hinton Agar (MHA) and place in the
incubator overnight. The number of colonies counted on the
overnight incubated plates divided by the plated volume and
multiplied by dilution factor yielded the actual number of
Klebsiella oxytoca bacteria recovered in CFU/mL.
[0105] The treated sample was diluted 10-fold and then the optical
density read was acquired at 625 nm to assess the amount of
particulate debris remaining in the sample.
Results
TABLE-US-00001 [0106] Pre-Treatment Optical Post-Treatment Optical
Fold Density (OD) Density (OD) Cleanup MEDIA 0.2841 0.065 4.35
METHOD
Electrode Configuration and Circuit Details
[0107] The 20 .mu.L of recovered sample volume was diluted with 40
.mu.L of 10 mM ascorbic acid and then introduced into a flow cell
(described below) for electrokinetic concentration.
[0108] For comparison purposes, a 20 .mu.L of a non-treated sample
was diluted with 40 .mu.L of 10 mM ascorbic acid and then
introduced into a flow cell (described below) for electrokinetic
concentration.
Flow Cell Construction
[0109] FIG. 4A is a perspective view of a multiple flow cell
laminate flow cell assembly 400, and FIG. 4B is a single flow cell
cutaway view with corresponding electrode and circuit details. Flow
cells were assembled using a three layer die-cut laminate flow cell
assembly 450 (DLE, Oceanside, Calif.), sandwiched between an indium
tin oxide (ITO) coated glass slide flow cell floor 451 (Delta
Technology, Stillwater, Minn.) and an ITO coated 5 mil polyester
(ITO PET) plastic flow cell ceiling 452 (Sheldahl, Northfield,
Minn.) forming a fluidic flow cell chamber. The laminate flow cell
assembly contained 32 separate channels 453, each having 1.78 mm
width.times.0.30 mm height.times.11.28 mm length, with 1.78 mm
diameter fluidic inlet and outlet ports (454 and 455, respectively)
to interface with plastic pipette tips for fluid exchanges using
manual pipettors. The transparent top and bottom electrodes enabled
microscope imaging.
[0110] Bacteria 401 suspended in redox active EKB were contacted
with uniform transparent electrodes constructed from transparent
ITO coated glass (Delta Technologies, Stillwater, Minn.) or
polyester film (Sheldahl, Northfield, Minn.). A potential was
applied to the conductive ITO surfaces completing the circuit,
establishing a faradaic current and an electric field between the
electrodes and enabling bacterial electrokinetic concentration
(EKC) and surface immobilization, as illustrated in FIG. 4B.
Bacterial Suspension and Surface Concentration Experiments
[0111] Studies were performed by loading the flow cells with
samples, with the power supply turned off, and then inserting the
flow cells onto the microscope stage. The microscope acquired
images at the bottom flow cell surface during subsequent steps. The
power supply was connected and cells electrokinetically
concentrated to the flow cell's bottom surface by application of a
1.4V DC fixed potential. The top electrode (flow cell ceiling) was
connected to the negative power supply terminal, and the bottom
electrode (flow cell floor) was connected to the positive terminal.
The applied potential resulted in complete bacterial concentration
in less than 3 minutes. After 300 seconds, a -1.0 V DC fixed
potential was applied for an additional 60 seconds to measure the
degree of irreversible binding of the sample debris and bacteria on
the flow cell floor. The digital microscope acquired images every
3-7 seconds during concentration.
Digital Microscopy Setup
[0112] An Olympus IX-71 inverted microscope equipped with a 12-bit,
1200.times.1600 pixel array monochrome CCD digital camera
(MicroFire, Leeds Precision Instruments, Minneapolis, Minn.) was
used for image acquisition. The transmitted illumination cone,
created with an IX-PH3 annular ring placed in a 0.55 NA transmitted
light condenser, was 33.4.degree. from the normal to the
microscope's focal plane. The illumination cone, after refraction
through the flow cell's air-glass-ITO-liquid interfaces (described
below), resulted in a 24.5.degree. forward scatter
angle-of-incidence relative to the focal plane normal. The forward
scatter angle-of-incidence relative to the flow cell's
air-plastic-ITO-liquid interfaces was not calculated. In all
formats, a dark image was obtained in the absence of scatterers, as
the illumination cone passed outside the 20.times., 0.4 NA
microscope objective's (LCPIanFI Olympus, Leeds Precision
Instruments) imaging cone. The presence of scatterers resulted in
the appearance of bright objects on a dark image background
(dark-field image for objects within the focal depth). The system
field-of-view was 444.times.592 .mu.m with corresponding 0.37 .mu.m
pixel resolution. The imaging system's depth-of-focus and image
depth were 5.8 .mu.m and 3.8 mm respectively. Constant camera
exposure and gain settings were maintained when relative intensity
comparisons were performed, as in the case of growth experiments
described below.
Accumulation Time Results
[0113] FIG. 5 is a graph showing results of accumulated objects
over time for treated compared to non-treated samples. The
non-treated sample data is expected data.
[0114] The objects are solid material, such as cells, viruses, and
cellular debris, that are immobilized on a sample surface. FIG. 5
shows that material concentrates, and then adheres to, the surface.
Subsequent processes, such as measuring the growth or growth rate,
can be utilized to determine the number of viable cells, and
additionally probing the material using receptor-ligand binding
techniques, including, but not limited to, antibody recognition or
nucleic acid hybridization methods can be used to measure the
abundance of microorganisms present.
[0115] FIGS. 6A, 6B and 6C depict microscopic images of non-treated
samples at initial time, time of 300 seconds, and time of 360
seconds, respectively. The surface accumulation rate is low. As
shown in FIGS. 6A, 6B and 6C, poor surface retention of the objects
occurs when samples are not treated.
[0116] FIGS. 7A, 7B and 7C depict microscopic images of treated
samples at initial time, time of 300 seconds, and time of 360
seconds, respectively. The treated sample surface accumulates all
objects, as evidenced by a plateau occurring in less than 3
minutes. As shown in FIGS. 7A, 7B and 7C, the objects were
irreversibly bound to the surface, as evidenced by consistent
accumulated counts during reverse electrophoresis.
Bacterial Growth
[0117] After cell immobilization, the flow cell was rinsed with 10
times the internal cell volume of 1/10th strength cation-adjusted
Mueller-Hinton Broth (CAMHB) growth media (Difco, Sparks, Md.). 100
.mu.L of liquefied Mueller Hinton Agar (MHA) was loaded into the
flow cell and then cooled to room temperature, solidifying into a
hydrogel.
Time Lapse Imaging
[0118] Direct observation of bacterial growth was performed by
inserting the disposable 32-channel flow cell assembly into a
custom benchtop automated instrument that combined digital
microscopy, motion control, and image analysis software. The system
was enclosed in an incubator maintained at 35.degree. C. The
motorized microscope stage enabled automated XY translation,
location logging, and memory with 10 .mu.m repeatability. The
system automatically focused and acquired surface images of
adherent bacteria at programmed time intervals for multiple
fields-of-view during an experiment. The system used the fiducial
markings to autofocus and mechanically align (.+-.1 pixel) the
fields-of-view prior to image acquisition. Unless stated otherwise,
a single field-of-view contained sufficient numbers of cells for
analysis, and automated analysis routines counted the number of
growing clones.
Growth Results
[0119] The number of growing clones observed using the digital
microscope method was compared with the number of expected growing
clones, assuming 100% yield and a 1 to 1 correlation between
growing colonies on MHA plates, to calculate a digital microscopy
method efficiency. The medium method was compared to an alternative
medium method wherein the gel was submerged. A total efficiency was
calculated by multiplying the treatment recovery and digital
microscopy efficiency.
TABLE-US-00002 Post Digital Treatment Microscopy Total Recovery EFF
EFF Control - No Prep 100% 12% 12% Medium Method 82% 90% 74%
Submerged Medium 43% 100% 43% Method
[0120] The total efficiency for the medium method was highest when
the gel slab was not submerged.
[0121] All directional references (e.g., proximal, distal, upper,
lower, upward, downward, left, right, lateral, front, back, top,
bottom, above, below, vertical, horizontal, clockwise, and
counterclockwise) are only used for identification purposes to aid
the reader's understanding of the present invention, and do not
create limitations, particularly as to the position, orientation,
or use of the invention. Connection references (e.g., attached,
coupled, connected, and joined) are to be construed broadly and may
include intermediate members between a collection of elements and
relative movement between elements unless otherwise indicated. As
such, connection references do not necessarily imply that two
elements are directly connected and in fixed relation to each
other. The exemplary drawings are for purposes of illustration
only, and the dimensions, positions, order and relative sizes
reflected in the drawings attached hereto may vary.
[0122] Numerous and varied other arrangements can be readily
devised by those skilled in the art without departing from the
spirit and scope of the description. Moreover, all statements
herein reciting principles, aspects and embodiments, as well as
specific examples thereof, are intended to encompass both
structural and functional equivalents thereof. Additionally, it is
intended that such equivalents include both currently known
equivalents as well as equivalents developed in the future, i.e.,
any elements developed that perform the same function, regardless
of structure. All references cited herein are incorporated by
reference in their entirety.
* * * * *